The inherent biological capacity for protein accumulation in developing crop seeds means that many crop plants, especially monocotyledonous, have the potential to be a practical and efficient vehicle for large-scale production of heterologous recombinant proteins, e.g. high-value polypeptides for the pharmaceutical industry; a manufacturing process often referred to as molecular farming. In addition, storing heterologous polypeptides in seeds reduces down-stream processing cost since these seeds may be stored for years without affecting the quality of the heterologous polypeptide. Expression of such proteins is, preferably, under the control of seed-specific or endosperm-specific promoters.
While the biotechnology industry continues on its path of producing pharmaceuticals and industrial proteins in food and/or feed crops, such as in cereals, there is a growing concern that seeds originating from transgenic plants may accidentally make their way into the human food supply through mixing of these seeds with non-transgenic seeds during harvesting, storage or down-stream processing. This is for example emphasized by the fact that seeds from transgenic cereals produced for molecular farming are non-distinguisable from non-transgenic seeds by simple visual inspection. Therefore, there is an urgent need in molecular farming for simple technology to distinguish transgenic seeds in the field or during harvesting and downstream processing from non-transgenic seeds for safeguarding the food supply and the environment. Furthermore, there is also an urgent need to accomplish this visual tracking without adding additional genes into the streamlined expression cassettes that may be used in molecular farming for high level expression of a particular high-value heterologous protein. Such technology would be an important factor in adopting strict guidelines for improved containment of transgenic seed material intended as raw matrial for the pharmaceutical industry. This is even more important when general guidelines for Good Agricultural Practices (GAP) are set forward as a prerequisite for all molecular farming of pharmaceuticals and industrials enzymes.
Tracing transgenic material non-distinguisable from non-transgenic material by simple visual inspection, such as seeds, can be very difficult and is both time-consuming and based on tedious extraction protocols and relatively expensive diagnostic technology such as quantitative PCR and real time PCR or ELISA.
Genetic markers such as screenable markers can be used in expression vectors to screen host organisms for effective transformation, such markers include a R-locus gene which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al. 1988), luciferase (lux) gene which allows for bioluminescence detection or a green fluorescent protein (GFP) gene. Although they may be useful in tissue culture following gene transfer none of these however are amenable for large-scale labeling and tracking in biofarming of GM crops because of e.g. difficulties in in situ application or because of requirements for a native biochemical precursor pathway to activate the marker that may be missing in the plant of interest.
It should also be noted that conventional transformation techniques employed in plant biotechnology frequently produce “chimeric” or “mosaic” hybrid lines that originate from more than one cell, and may result in plants expressing a heterologous gene of interest introduced in the plant but not the screenable marker. Expression of screenable markers introduced by expression vectors in transformation frequently dissappears between generations and such markers may therefore be unreliable traits for visual inspection.
With increased general safety concerns relating to genetically modified crops and biofarming there is urgent need for safe and effective methods for producing genetically modified plants that are safely contained and reliably distinguishable from non-modified wild or farmed plants.